A better understanding of the molecular basis of both DNA double strand break (DSB) repair, and the immediate responses of human breast cancer cells to DNA damage after ionizing radiation (IR), which are important determinants in resistance, cell death or survival, will be required to improve therapy of advanced disease. Recently, the principal investigator's laboratory has isolated X-ray-inducible transcript-8 (xip8) [i.e., testosterone repressed message-2 (TRPM-2)/apolipoprotein J (ApoJ)], a protein that strongly associate with the Ku780 DSB repair protein. It is hypothesized that the induction and overexpression of the nuclear form of XIP8/TRPM-2/ApoJ after IR inhibits Ku70/Ku8O DNA end-binding, ATPase and/or helicase functions, resulting in loss of DNA- PK activity and nonhomologous DSB repair. XIP8 is a key determinant in apoptosis/survival responses in severely damaged human breast cancer (e.g., MCF-7) cells. The applicant will test this hypothesis by: (a) examining repair apoptotic and survival responses of MCF-7 cells that overexpress the nuclear form of XIP8, or in cells that are deficient in its expression (using cells from XIP8/ApoJ knockout mice) before and after IR (Years 0-5); (b) examining the effects of the nuclear form of XIP8 on Ku70-Ku-80 protein-protein interactions and DSB repair (Years 0-5); (c) elucidating the protein-protein interaction domains between XIP8 and Ku70 using yeast two-hybrid and co-immunoprecipitation analysis (Years 0-3); and (d) performing structure/function analysis of XIP8 in cells and cell lines from ApoJ/XIP8 knockout mice using Ku70/Ku8O activity assays and Ku70-XIP8 protein-protein interactions (Years 0-3). Dominant-negative versions of XIP8 and Ku70 will be constructed-for use as molecular radiosensitizers (Years 2-5). These Specific Aims will directly test the hypothesis that the nuclear form of XIP8 is a determining factor in blocking nonhomologous DSB repair and regulating apoptosis when over-expressed in the nucleus of damaged cells. We will also determine if XIP8 induction plays a prominent role in cell cycle checkpoint regulation after IR.